Malaria sporozoites actively enter and pass through rat Kupffer cells prior to hepatocyte invasion

Abstract

Malaria sporozoites have to cross the layer of sinusoidal liver cells to reach their initial site of multiplication in the mammalian host, the hepatocytes. To determine the sinusoidal cell type sporozoites use for extravasation, endothelia or Kupffer cells, we quantified sporozoite adhesion to and invasion of sinusoidal cells isolated from rat liver. In vitro invasion assays reveal that Plasmodium berghei and P. yoelii sporozoites attach to and enter Kupffer cells, but not sinusoidal endothelia. Unlike hepatocytes and other nonphagocytic cells, which are invaded in vitro only within the first hour of parasite exposure, the number of intracellular sporozoites in Kupffer cells increases for up to 12 hours. By confocal and electron microscopy, sporozoites are enclosed in a vacuole that does not colocalize with lysosomal markers. Inhibition of phagocytosis with gadolinium chloride has no effect on Kupffer cell invasion, but abolishes phagocytosis of inactivated sporozoites. Malaria-infected anopheline mosquitoes inoculate Plasmodium sporozoites into the avascular dermis and subcutaneous connective tissue of the mammalian host. 1 Within minutes after entering a blood capillary at the mosquito bite site, the sporozoites travel in the bloodstream to the liver where they invade their initial target cells, the hepatocytes. 2 The arrest of the sporozoites in the liver sinusoid is thought to be mediated by the specific binding of the major Plasmodium sporozoite surface proteins, the circumsporozoite (CS) protein and the thrombospondin-related adhesive protein, to the unique heparan sulfate proteoglycans (HSPGs) of the liver. 3-5 For example, recombinant CS protein binds to the HSPGs on the basolateral cell surface of hepatocytes in the space of Disse. This interaction occurs between the conserved regions I and II-plus of the CS protein and highly sulfated, heparin-like oligosaccharides in heparan sulfate. 6 Furthermore, recombinant CS protein is selectively removed from the bloodstream by binding to liver HSPGs, 3,6,7 a clearance mechanism also used by lipoprotein remnants. [8][9][10][11] Since P. berghei sporozoites compete with ␤ very low density lipoprotein for binding to HSPGs, 12 it appears that malaria sporozoites utilize an existing clearance mechanism of the host for targeting the liver sinusoid.After the initial arrest in the liver sinusoid, Plasmodium sporozoites have to cross the sinusoidal cell layer prior to invasion of hepatocytes. The sinusoid is composed of fenestrated endothelial cells and interspersed with Kupffer cells. Kupffer cells are resident macrophages of the liver that are strategically positioned in the sinusoidal lumen and exhibit numerous pseudopodia. To date, the route that sporozoites use for extravasation has remained uncertain. [13][14][15] have proposed that the parasites infect hepatocytes directly by squeezing through the endothelial fenestration, although they exceed the maximum pore size of the sieve plates (0.1 m 16 ) by an order of magnitude. However, this event has never been observ...

A long and winding road: The Plasmodium sporozoite's journey in the mammalian host

“…To cross the sinusoid, sporozoites may migrate through or between the endothelial cells [46] or they may use Kupffer cells as a gateway to the underlying hepatocyte, a hypothesis put forward after sporozoites were observed inside Kupffer cells at early time points after injection [47]. More recent in vitro observations of the interaction between sporozoites and sinsusoidal cells found that sporozoites preferentially associate with Kupffer cells and are capable of going through Kupffer cells to productively enter hepatocytes [48]. In addition, op/op mice which have reduced numbers of Kupffer cells have somewhat lower parasite burdens when inoculated with sporozoites by mosquito bite [49].…”

“…In the normal hepatic sinusoid, however, the majority of sporozoites probably gain access to the hepatic parenchyma by crossing Kupffer cells. Controversy remains as to how Kupffer cells are crossed, whether it is by a unique mechanism involving parasitophorous vacuole formation [48] or whether they traverse these cells in the same way they traverse other cells, a hypothesis suggested by the inability of SPECT 1, SPECT 2 and CelTOS knockout sporozoites to cross this barrier.…”

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“…Kupffer cells are specialized macrophages and protect the liver against microbes, contaminants and other aggressions. Why these phagocytes are used as entry gate by sporozoites indeed is difficult to understand [12]. But some early studies have shown that IgA antibodies preferentially attach to hepatocytes, blocking the entry for sporozoites.…”

In vivo imaging of malaria parasites in the murine liver

“…Most of our views on how the parasite goes into and out of the hepatic tissue come from transmission electron microscopy approaches, whose significance is frequently limited by the small number of events reported, or from in vitro studies, which have their own limitations [7][8][9] . In recent years, imaging technology has been transferred from physics laboratories to the biological domain with impressive efficiency, which has revolutionized the way in vivo questions can be tackled 10,11 .…”